Protein tyrosine kinase enzyme inhibitors

ABSTRACT

This invention provides compounds of formula 1, having the structure  
                 
 
wherein R 1 , R 2 , R 3 , R 4 , and R 5  are described within the specification.

This application claims priority from copending provisional applicationSer. No. 60/560,724, filed Sep. 15, 2003, converted from nonprovisionalapplication Ser. No. 10/662,273, filed Sep. 15, 2003, the entiredisclosure of which is hereby incorporated by reference.

BACKGROUND OF THE INVENTION

This invention relates to certain substituted 3-cyano quinolinecompounds as well as the pharmaceutically acceptable salts thereof. Thecompounds of the present invention inhibit the HER-2 and epidermalgrowth factor receptor (EGFR) enzyme thereby inhibiting the abnormalgrowth of certain cell types. The compounds of this invention areanti-cancer agents and are useful for the treatment of cancer inmammals. This invention also relates to the use of 3-cyano quinolines inthe treatment of cancer and the pharmaceutical preparations containingthem.

Protein tyrosine kinases are a class of enzymes that catalyze thetransfer of a phosphate group from ATP to a tyrosine residue located ona protein substrate. Protein tyrosine kinases clearly play a role innormal cell growth. Many of the growth factor receptor proteins functionas tyrosine kinases and it is by this process that they effectsignaling. The interaction of growth factors with these receptors is anecessary event in normal regulation of cell growth. However, undercertain conditions, as a result of either mutation or over expression,these receptors can become deregulated, the result of which isuncontrolled cell proliferation which can lead to tumor growth andultimately to the disease known as cancer [Walks, A. F., Adv. CancerRes., 60, 43 (1993) and Parsons, J. T.; Parsons, S. J., ImportantAdvances in Oncology, DeVita, V. T. Ed., J. B. Lippincott Co., Phila, 3(1993)]. Among the growth factor receptor kinases and theirproto-oncogenes that have been identified and which are targets of thecompounds of this invention are the epidermal growth factor receptorkinase (EGF-R kinase, the protein product of the erbB oncogene), and theproduct produced by the erbB-2 (also referred to as the neu or HER-2)oncogene. Since the phosphorylation event is a necessary signal for celldivision to occur and since over expressed or mutated kinases have beenassociated with cancer, an inhibitor of this event, a protein tyrosinekinase inhibitor, will have therapeutic value for the treatment ofcancer and other diseases characterized by uncontrolled or abnormal cellgrowth. For example, over expression of the receptor kinase product ofthe erbB-2 oncogene has been associated with human breast and ovariancancers [Slamon, D. J., et. al., Science, 244, 707 (1989) and Science,235, 1146 (1987)]. Deregulation of EGF-R kinase has been associated withepidermoid tumors [Reiss, M., et al., Cancer Res., 51, 6254 (1991)],breast tumors [Macias, A., et. al., Anticancer Res., 7, 459 (1987)], andtumors involving other major organs [Gullick, W. J., Brit. Med. Bull.,47, 87 (1991)]. Because of the importance of the role played byderegulated receptor kinases in the pathogenesis of cancer, many recentstudies have dealt with the development of specific PTK inhibitors aspotential anti-cancer therapeutic agents [some recent reviews: Burke. T.R., Drugs Future, 17, 119 (1992) and Chang, C. J.; Geahlen, R₁ L., J.Nat. Prod., 55, 1529 (1992)]. The compounds of this invention inhibitthe kinase activity of EGF-R and are therefore useful for treatingcertain disease states, such as cancer, that result, at least in part,from deregulation of this receptor.

The HER-2 gene (c-erbB-2, neu) encodes a 185 kDa transmembrane tyrosinekinase receptor that has partial homology with other members of theepidermal growth factor receptor family [Shih, C., Padhy, L. C., Murray,M., et al. Transforming genes of carcinomas and neuroblastomasintroduced into mouse fibroblasts, Nature, 290, 261-264 (1981)]. It isnow known that normal human cells express a small constitutive amount ofHER-2 protein on the plasma membrane. The activation of the HER-2oncogene is believed to follow the binding of a yet unidentified growthfactor ligand to the HER-2 receptor complex, which leads toheterodimerization, triggering a cascade of growth signals thatculminates in gene activation. More specifically, the epidermal growthfactor family can be subdivided into four groups based on theirreceptor-binding specificities (HER-1, HER-2, HER-3, and HER-4). HER-2is the preferred heterodimerization partner of all other HER receptors.Over expression of HER-2 has been demonstrated to lead to increasedtumorigenicity, tumor invasiveness, increased metastatic potential, andaltered sensitivity to hormonal and chemotherapeutic agents intransfection studies in cellular and animal models [Pegram, M. D., Finn,R₁ S., Arzoo, K., et al. The effect of HER-2/neu over expression onchemotherapeutic drug sensitivity in human breast and ovarian cellsOncogene, 15, 537-547 (1997)].

HER-2 protein over expression has been reported to occur inapproximately 30% of invasive human breast cancers, with HER-2 geneamplification detected in 95% or more of the specimens found to overexpress HER-2 protein, [Gebhardt, F., Zanker, K., Brandt, B.Differential expression of alternatively spliced c-erbB-2 mRNA inprimary tumors, lymph node metastases, and bone marrow micro metastasesfrom breast cancer patients Biochem. Biophys. Res. Commun., 247, 319-323(1998)].

U.S. Pat. No. 6,288,082 issued Sep. 11, 2001 (the '082 patent) disclosessubstituted 3-cyano quinoline compounds that inhibit epidermal growthfactor receptor (EGFR). The compounds of this application aredistinguished from those of the '082 patent in their ability to act aspotent HER-2 inhibitors.

BRIEF SUMMARY OF THE INVENTION

This invention provides a compound of formula 1:

wherein:

-   R₁ is halogen;-   R₂ is a pyridinyl, thiophene, pyrimidine, thiazole, or phenyl    optionally substituted with up to three substituents;-   R₃ is —O— or —S—;-   R₄ is methyl or CH₂CH₂OCH₃;-   R₅ is ethyl or methyl; and-   n is 0 or 1.

In one embodiment the compounds of this invention include:

-   (E)-N-{4-[4-(benzyloxy)-3-chloroanilino]-3-cyano-7-ethoxy-6-quinolinyl}-4-(dimethylamino)-2-butenamide;-   (E)-N-{4-[3-chloro-4-(2-pyridinylmethoxy)anilino]-3-cyano-7-ethoxy-6-quinolinyl}-4-(dimethylamino)-2-butenamide;-   (E)-N-(4-{3-chloro-4-[(3-fluorobenzyl)oxy]anilino}-3-cyano-7-ethoxy-6-quinolinyl)-4-(dimethylamino)-2-butenamide;-   (2E)-N-(4-{[3-chloro-4-(1,3-thiazol-2-ylsulfanyl)phenyl]amino}-3-cyano-7-methoxy-6-quinolinyl)-4-(dimethylamino)-2-butenamide;-   (E)-N-(4-{3-chloro-4-[(4,6-di-methyl-2-pyrimidinyl)sulfanyl]anilino}-3-cyano-7-ethoxy-6-quinolinyl)-4-(dimethylamino)-2-butenamide;-   (E)-N-{4-[3-chloro-4-(1,3-thiazol-2-ylsulfanyl)anilino]-3-cyano-7-methoxy-6-quinolinyl}-4-[(2-methoxyethyl)(methyl)amino]-2-butenamide;    -   The following experimental details are set forth to aid in an        understanding of the invention, and are not intended, and should        not be construed, to limit in any way the invention set forth in        the claims that follow thereafter.

DETAILED DESCRIPTION OF THE INVENTION

The compounds of this invention are certain substituted 3-cyanoquinolines. Throughout this patent application, the quinoline ringsystem will be numbered as indicated in the formula below; the numberingfor the quinazoline ring system is also shown:

The pharmaceutically acceptable salts of the compounds of this inventionare those derived from such organic and inorganic acids as: acetic,lactic, citric, tartaric, succinic, maleic, malonic, gluconic,hydrochloric, hydrobromic, phosphoric, nitric, sulfuric,methanesulfonic, and similarly known acceptable acids.

For purposes of this invention “halogen” is F, Cl, Br, or I.

Where a group is referred to as “substituted”, preferred substituentsare selected from alkyl of up to six carbon atoms, alkoxy, of up to sixcarbon atoms and halogen. A particularly preferred substituent ismethyl.

The compounds of this invention may contain one or more asymmetriccarbons atoms; in such cases, the compounds of this invention includethe individual diasteromers, the racemates, and the individual R and Sentantiomers thereof. Some of the compounds of this invention maycontain one or more double bonds; in such cases, the compounds of thisinvention include each of the possible configurational isomers as wellas mixtures of these isomers.

For purposes of this invention a “neoplasm” is defined as cells selectedfrom the breast, kidney, bladder, mouth, larynx, esophagus, stomach,colon, ovary, pancreas, brain, prostrate and lung having a morphologynot found in the majority of the cells of a mammal.

In one embodiment, the present invention provides for a method ofinhibiting the neoplasm. The method comprises contacting a cell with anamount of a compound effective to decrease or prevent HER-2 function.The cell may be a mammalian cell and more specifically a human cell. Thecell may also be a bacterial cell such as for example E. Coli. The cellmay include but is not limited to, a neuronal cell, an endothelial cell,a glial cell, a microglial cell, a smooth muscle cell, a somatic cell, abone marrow cell, a liver cell, an intestinal cell, a germ cell, amyocyte, a mononuclear phagocyte, an endothelial cell, a tumor cell, alymphocyte cell, a mesangial cell, a retinal epithelial cell, a retinalvascular cell, a ganglion cell or a stem cell. The cell may be a normalcell, an activated cell, a neoplastic cell, a diseased cell, or aninfected cell.

In another embodiment, the present invention provides a method for thetreatment or prevention of a neoplasm in a mammal. The present inventionaccordingly provides to a mammal, a pharmaceutical composition thatcomprises a compound of this invention in combination or associationwith a pharmaceutically acceptable carrier. The compound of thisinvention may be administered alone or in combination with othertherapeutically effective compounds or therapies for the treatment orprevention of the neoplasm.

The compounds may be provided orally, by intralesional, intraperitoneal,intramuscular or intravenous injection; infusion; liposome-mediateddelivery; topical, nasal, anal, vaginal, sublingual, uretheral,transdermal, intrathecal, ocular or otic delivery. In order to obtainconsistency in providing the compound of this invention it is preferredthat a compound of the invention is in the form of a unit dose. Suitableunit dose forms include tablets, capsules and powders in sachets orvials. Such unit dose forms may contain from 0.1 to 300 mg of a compoundof the invention and preferably from 2 to 100 mg. Still furtherpreferred unit dosage forms contain 5 to 50 mg of a compound of thepresent invention. The compounds of the present invention can beadministered orally at a dose range of about 0.01 to 100 mg/kg orpreferably at a dose range of 0.1 to 10 mg/kg. Such compounds may beadministered from 1 to 6 times a day, more usually from 1 to 4 times aday. The effective amount will be known to one of skill in the art; itwill also be dependent upon the form of the compound. One of skill inthe art could routinely perform empirical activity tests to determinethe bioactivity of the compound in bioassays and thus determine whatdosage to administer.

The compounds of the invention may be formulated with conventionalexcipients, such as a filler, a disintegrating agent, a binder, alubricant, a flavoring agent, a color additive, or a carrier. Thecarrier may be for example a diluent, an aerosol, a topical carrier, anaqueous solution, a nonaqueous solution or a solid carrier. The carriermay be a polymer or a toothpaste. A carrier in this inventionencompasses any of the standard pharmaceutically accepted carriers, suchas phosphate buffered saline solution, acetate buffered saline solution,water, emulsions such as an oil/water emulsion or a triglycerideemulsion, various types of wetting agents, tablets, coated tablets andcapsules.

When provided orally or topically, such compounds would be provided to asubject by delivery in different carriers. Typically, such carrierscontain excipients such as starch, milk, sugar, certain types of clay,gelatin, stearic acid, talc, vegetable fats or oils, gums, or glycols.The specific carrier would need to be selected based upon the desiredmethod of delivery, for example, phosphate buffered saline (PBS) couldbe used for intravenous or systemic delivery and vegetable fats, creams,salves, ointments or gels may be used for topical delivery.

The compounds of the present invention may be delivered together withsuitable diluents, preservatives, solubilizers, emulsifiers, adjuvantsand/or carriers useful in treatment or prevention of neoplasm. Suchcompositions are liquids or lyophilized or otherwise dried formulationsand include diluents of various buffer content (for example, Tris-HCl,acetate, phosphate), pH and ionic strength, additives such as albuminsor gelatin to prevent absorption to surfaces, detergents (for example,TWEEN 20, TWEEN 80, PLURONIC F68, bile acid salts), solubilizing agents(for example, glycerol, polyethylene glycerol), anti-oxidants (forexample ascorbic acid, sodium metabisulfate), preservatives (forexample, thimerosal, benzyl alcohol, parabens), bulking substances ortonicity modifiers (for example, lactose, mannitol), covalent attachmentof polymers such as polyethylene glycol, complexation with metal ions,or incorporation of the compound into or onto particulate preparationsof hydrogels or liposomes, micro-emulsions, micelles, unilamellar ormultilamellar vesicles, erythrocyte ghosts, or spheroblasts. Suchcompositions will influence the physical state, solubility, stability,rate of in vivo release, and rate of in vivo clearance of the compoundor composition. The choice of compositions will depend on the physicaland chemical properties of the compound capable of treating orpreventing a neoplasm.

The compound of the present invention may be delivered locally via acapsule that allows a sustained release of the compound over a period oftime. Controlled or sustained release compositions include formulationin lipophilic depots (for example, fatty acids, waxes, oils).

The present invention further provides a compound of the invention foruse as an active therapeutic substance for preventing neoplasm.

The present invention further provides a method of treating neoplasm inhumans, which comprises administering to the infected individual aneffective amount of a compound or a pharmaceutical composition of theinvention.

The compounds of this invention can be prepared as outlined in Flowsheet1 wherein R₁, R₂, and R₃ are as described above. The amino group ofcompound 1 can be protected as an amide group by acetylation usingacetic anhydride in a solvent such as acetic acid. The hydroxyl group of2 can be alkylated with an alkyl bromide, iodide, tosylate, or mesylateusing potassium carbonate in a refluxing solvent such as acetone. Thenitro group of 3 can be reduced using catalytic hydrogenation to givethe substituted aniline 4. Heating of 4 with reagent 5 with or without asolvent gives the intermediate 6. Refluxing 6 in a high boiling solventsuch as Dowtherm results in cyclization to the hydroxy quinoline 7. Thiscan be chlorinated by heating in phosphorous oxychloride to give thechloro derivative 8. Condensation of 8 with an aniline of formula 9 in arefluxing solvent such as ethanol in the presence of a catalytic amountof acid yields the intermediate 10. The acetate group of 10 can beremoved by hydrolysis using acidic or basic conditions followed byneutraliztion to give 11. The intermediate 11 can be acylated with anamino acid chloride 12 (as the hydrochloride salt) to give the compoundsof this invention of formula 13. Methods used to prepare the compoundsin U.S. Pat. No. 6,288,082, WO-9633978 and WO-9633980 can also be usedto prepare the compounds of this invention and are hereby incorporatedby reference.

In addition to the method described herein above, there a number ofpatent applications that describe methods that are useful for thepreparation of the compounds of this invention. Although these methodsdescribe the preparation of certain quinazolines, they are alsoapplicable to the preparation of correspondingly substituted3-cyanoquinolines and are hereby incorporated by reference. The chemicalprocedures described in the application WO-9633980 can be used toprepare the 3-cyanoquinoline intermediates used in this inventionwherein the substitution at position 6 is an aminoalkylalkoxy group. Thechemical procedures described in the application WO-9633978 can be usedto prepare the 3-cyanoquinoline intermediates used in this inventionwherein the substitution at position 6 is an aminoalkylamino group.

EXAMPLE 1

(E)-N-{4-[4-(benzyloxy)-3-chloroanilino]-3-cyano-7-ethoxy-6-quinolinyl}-4-(dimethylamino)-2-butenamide

A 1.74 ml (2.54 g, 0.02 moles) portion of oxalyl chloride was added to3.31 grams (0.02 moles) of (E)-4-(dimethylamino)-2-butenoic acidhydrochloride in 75 ml of acetonitrile. To this was added a small dropof dimethylformamide. The reaction was heated and stirred in an oil bathat 63° for 20 minutes, giving an orange solution. This solution asconcentrated in vacuo without the application of heat to about half itsoriginal volume. This solution was cooled in an ice bath and a solutionof 4.45 g (0.01 moles) of the6-amino-4-[4-(benzyloxy)-3-chloroanilino]-7-ethoxy-3-quinolinecarbonitrilein 50 ml of N-methylpyrrolidone was added in a stream. The reaction wascooled and stirred for 2 hours. The reaction was poured onto 100 ml ofsaturated aqueous sodium bicarbonate in ice. On standing the resultinggum solidified and the solid was filtered. This solid waschromatographed on silica gel. The column was washed with 3 liters of1:19 methanol-ethyl acetate, then the product was eluted with 3 litersof 1:5:94 triethylamine-methanol-ethyl acetate. Concentration of theeluate gave a solid, which was filtered to give 2.96 grams of the titlecompound. From the filtrate was obtained an additional 1.0 grams ofproduct. Total: 3.96 g.

EXAMPLE 2(E)-N-{4-[3-chloro-4-(2-pyridinylmethoxy)anilino]-3-cyano-7-ethoxy-6-quinolinyl}-4-(dimethylamino)-2-butenamide

A solution of (E)-4-(dimethylamino)-2-butenoic acid hydrochloride in 1.2L of tetrahydrofuran (THF) and a catalytic amount of dimethylformide(DMF) (1.2 ml) was cooled to 0-5° C. Oxalyl chloride (0.95 eq) was addeddropwise and the mixture was warmed to 25-30° C. and stirred for 2hours. The orange suspension was checked for complete consumption ofoxalyl chloride by HPLC then cooled to 0-5° C. A solution of 111 g of4-[4-(2-pyridylmethoxy)-3-chloro]amino-6-amino-3-cyano-7-ethoxyquinolinein 1.47 L of 1-methyl-2-pyrrolidinone was added dropwise and the mixturewas stirred until ≦1.0% of the starting aniline remained (3-16 hours).The reaction was quenched with water and the mixture was warmed to 40°C. Aqueous sodium hydroxide was added to bring the pH to 10-11. Theresulting precipitates were filtered hot and washed with water. The wetsolids were heated to reflux (70-75° C.) in acetonitrile:THF (1.5:1) andthe solution cooled over 3 hours to room temperature. The product wasfiltered and washed with acetonitrile:THF. The product was dried (50°C., 10 mm Hg, 24 hours) to give 80-85% yield. Melting point of maleatesalt 178-183° C.

EXAMPLE 3(E)-N-(4-{3-chloro-4-[3-fluorobenzyl)oxy]anilino}-3-cyano-7-ethoxy-6-quinolinyl)-4-(dimethylamino)-2-butenamide

A solution of 108 g (E)-4-(dimethylamino)-2-butenoic acid hydrochloridein 1.1 L of tetrahydrofuran (THF) and a catalytic amount ofdimethylformide (DMF) (1.2 ml) was cooled to 0-5° C. Oxalyl chloride(0.95 eq) was added dropwise and the mixture was warmed to 25-30° C. andstirred for 2 hours. The orange suspension was checked for completeconsumption of oxalyl chloride by HPLC then cooled to 0-5° C. A solutionof 150 g of6-amino-4-[3-chloro-4-(3-fluorobenzyloxy)]anilino-3-cyano-7-ethoxyquinoline in 1.5 L of 1-methyl-2-pyrrolidinone was added dropwise andthe mixture was stirred until ≦1.0% of the starting aniline remained(3-16 hours). The reaction was quenched with water and the mixture waswarmed to 40° C. Aqueous sodium hydroxide (101 g in 750 ml) was added tobring the pH to 10-11. The resulting precipitates were filtered hot andwashed with water. The wet solids were heated to reflux (70-75° C.) inacetonitrile:THF (1.5:1) and the solution was cooled over 3 hours toroom temperature. The product was filtered and washed withacetonitrile:THF. The product was dried (50° C., 10 mm Hg, 24 h) andobtained in 80-85% yield. Melting Point 165-167° C.

EXAMPLE 4 4-Benzyloxy-3-chloro-nitrobenzene

A 15.43 g (0.275 moles) portion of solid (pellets) potassium hydroxidewas added to a solution of 43.89 g (0.25 moles) of 3-chloro-4-fluoronitrobenzene and 32.34 ml (33.79 grams, 0.373 moles) of benzyl alcoholin 220 ml of acetonitrile. The reaction was vigorously stirred with amechanical stirrer overnight. The resulting solid was filtered.Concentration of the filtrate gave a second crop, which was alsofiltered. On standing more solid came out of this filtrate. This mixturewas treated with ether, and the solid filtered. All solids were washedthoroughly with water, and combined to give 49.71 g of the titlecompound.

EXAMPLE 5 4-Benzyloxy-3-chloro-phenylamine

A mixture of 6.59 g (0.025 moles) of the 4-benzyloxy-3-chloronitrobenzene (example 4), 4.19 g (0.075 moles) of iron powder, and 12.04g (0.225 moles) of ammonium chloride in 100 ml of ethanol and 25 ml ofwater was stirred mechanically and refluxed for half an hour. Thereaction was allowed to cool and stir for 1 hour. The mixture wasfiltered and solids were washed with ethanol. The combined filtrateswere taken to dryness in vacuo. This solid was dissolved in methylenechloride and passed through Magnesol. Removal of the solvent from thefiltrate in vacuo gave 5.60 g of title compound.

EXAMPLE 6N-{4-[4-(Benzyloxy)-3-chloroanilino]-3-cyano-7-ethoxy-6-quinolinyl}acetamide

A mixture of 4.17 g (0.0149 moles) of theN-(4-chloro-3-cyano-7-ethoxy-6-quinolinyl)acetamide, 4.04 g (0.0173moles) of 4-benzyloxy-3-chloro-phenylamine (example 5), and 2.0 g (0.017moles) of pyridine hydrochloride in 85 ml of isopropanol was stirred andrefluxed in an oil bath for 30 minutes. The reaction was cooled in anice bath, and the solid was collected by filtration and washed withisopropanol, and then with ether yielding 7.26 g of crude product as thehydrochloride salt. This material was purified by chromatography of thefree base on silica gel by elution with 1:39 methanol-methylenechloride.

EXAMPLE 76-Amino-4-[4-(benzyloxy)-3-chloroanilino]-7-ethoxy-3-quinolinecarbonitrile

A solution of 298 mg (0.612 mmoles) of the purifiedN-{4-[4-(benzyloxy)-3-chloroanilino]-3-cyano-7-ethoxy-6-quinolinyl}acetamide(example 6) and 97 mg (1.73 mmoles) of potassium hydroxide in 10 ml ofmethanol was stirred and refluxed for 60 hours. On cooling a solidformed. This mixture was poured onto ice, and the resulting solid wasfiltered and washed with water. On drying, 242 mg of the title compoundwas obtained.

EXAMPLE 8 2-Acetamido-5-nitrophenol

To 400 g of 2-amino-5-nitrophenol in a 5-L multi-necked flask equippedwith a mechanical stirrer, reflux condenser, nitrogen inlet, 500-mLaddition funnel, heating mantle, and a thermocouple attached to atemperature controller was added 1.6 L of acetic acid. The mixture wasstirred at 60° C. as 398 g of acetic anhydride was added over 1.5 hours.After 1 hour, another 37 g of acetic anhydride was added. After another1 hour, the mixture was cooled and diluted with 2 L of water. Solid wascollected by filtration and washed with water and heptane. The solid wasdried in a vacuum oven to give 509 g of the title compound.

EXAMPLE 9 4-Acetamido-3-ethoxynitrobenzene

To 400 g of 2-acetamido-5-nitrophenol in a 12-L, 4-necked flask equippedwith a reflux condenser, nitrogen inlet, thermocouple, addition funnel,and mechanical stirrer was added 790 g of potassium carbonate and 2.0 Lof dimethylformamide. The mixture was stirred at 60° C. as 294 g ofethyl bromide was added over 30 minutes. After 1 hour, an additional 27g of ethyl bromide was added and the mixture was stirred at 60° C. foranother hour. The mixture was cooled to room temperature and poured into4 L of water. After 30 minutes, the product was collected by filtrationand washed with water and heptane. The product was dried in a vacuumoven at 60° C. to give 457 g of the title compound.

EXAMPLE 10 3-(4-Acetamido-3-ethoxyaniline)-2-cyanopropenoic acid ethylester

A suspension of 4-acetamido-3-ethoxynitrobenzene compound intetrahydrofuran (10 parts) was reduced to the aniline derivative using10% Pd/C wet at 50 psi hydrogen and 30° C. for 2 hours. The resultingsolution was filtered and concentrated to 2 parts of tetrahydrofuran.The concentrate was diluted with toluene and allowed to react withcommercially available ethyl (ethoxymethylene)cyanoacetate at reflux for16 hours. After reaction completion, the mixture was cooled. Theprecipitated product was collected by filtration, washed and dried. Theproduct was obtained in 90% yield.

EXAMPLE 11 3-Cyano-7-ethoxy-4-hydroxy-6-N-acetylquinoline

A solution of 210 g of 3-(4-acetamido-3-ethoxyaniline)-2-cyanopropenoicacid ethyl ester in 12 L of Dowtherm was stirred under nitrogen at 250°C. for 15 to 20 hours. The mixture was cooled to room temperature andsolid was collected by filtration. The solid was washed with toluene andmixed with 1.2 L of tetrahydrofuran. The mixture was refluxed for 30minutes and then cooled to room temperature. The solid was collected andwashed with tetrahydrofuran. After drying 179.4 g of the title compoundwas obtained.

EXAMPLE 12 4-Chloro-3-cyano-7-ethoxy-6-nitro quinoline

A stirred mixture of 300 g3-cyano-7-ethoxy-4-hydroxy-6-N-acetylquinoline in 2.53 L of1,2-diethoxyethane was heated to 80-85° C. To this was added 224 ml ofphosphorus oxychloride over 30-40 minutes. The mixture was stirred at80-85° C. for 2-4 hours. The mixture was cooled, filtered over a celitepad and washed with 1,2-diethoxyethane. The filtrates were added over1.5 hours to a cooled (0-10° C.) potassium carbonate (537 g in 1.5 Lwater) solution. The resulting yellow mixture was stirred for a minimumof 12 hours. The mixture was filtered and washed with hot water. Thesolids were dried (50° C., 10 mm Hg, 24 h) to give the title compound in30-50% yield. The material was used directly in the next step.

EXAMPLE 13 3-Chloro-4-(2-pyridylmethoxy)nitrobenzene

A mixture of 160 g of potassium hydroxide and 2-pyridylcarbinol in 8 Lacetonitrile was stirred for 20-30 minutes. To this was added 400 g of3-chloro-4-fluoronitrobenzene and the mixture was stirred at 40° C. fora minimum of 18 hours until the reaction was complete. Water was addedand the precipitated yellow solids were filtered and washed with water.The product was dried (40-50° C., 10 mm Hg, 24 h) to the product in85-95% yield.

EXAMPLE 14 3-Chloro-4-(3-fluorobenzyloxy)nitrobenzene

This compound was prepared from 3-chloro-4-fluoronitrobenzene and3-fluorobenzyl alcohol using the method described above in Example 13.

EXAMPLE 156-Amino-4-(3-chloro-4-(3-fluorobenzyloxy))anilino-3-cyano-7-ethoxyquinoline

To a mixture of 400 g of 3-chloro-4-(3-fluorobenzyloxy)nitrobenzene(example 14) and 464 g zinc dust in 4 L of ethanol at 40-50° C. wasadded aqueous ammonium chloride (152 g in 800 ml water). After stirringa minimum of 2 hours, the reaction mixture was filtered hot through acelite pad and washed with hot ethanol. The filtrate was evaporated and1.72 L of 2-methyl THF, water and brine were added. The organic layerwas separated and washed with water. The organic layer was evaporatedand replaced with 3.8 L of ethanol.4-Chloro-3-cyano-7-ethoxy-6-N-acetylamino-quinoline was added with acatalytic amount of methane sulfonic acid and the mixture was heated at70-75° C. for a minimum of 2 hours until reaction completion.Concentrated 1.69 L HCl was added at 70-75° C. and held for a minimum of2 hours until complete hydrolysis. Water was added and the mixture wascooled to 40° C., solid was collected and washed with water. The wetcake was slurried in 5.4 L of methanol, 10% aqueous potassium carbonate(315 g in 2.8 L water) was added and the mixture was stirred for 2.5hours. The mixture was filtered and washed with 1:1 methanol:water. Theproduct was dried (50° C., 10 mm Hg, 24 hours) to give the titlecompound in 80-90% yield.

EXAMPLE 166-Amino-4-(4-(2-pyridylmethoxy)-3-chloro)anilino-3-cyano-7-ethoxyquinoline

The above-identified compound was prepared from3-chloro-4-(2-pyridylmethoxy)nitrobenzene and4-chloro-3-cyano-7-ethoxy-6-N-acetylamino-quinoline using the methoddescribed above in Example 15.

EXAMPLE 20 4-Dimethyl-but-2-enoic acid[4-(3-chloro-4-fluoro-phenylamino)-3-cyano-7-ethoxy-quinolin-6-yl]-amideEXAMPLE 21N-{4-[3-Chloro-4-(1-methyl-1H-imidazol-2-ylsulfanyl)-phenylamino]-3-cyano-quinolin-6-yl}-acrylamideEXAMPLE 22 6,7-Diethoxy-4-(1H-indol-6-ylamino)-quinoline-3-carbonitrileEXAMPLE 234-(2,3-Dihydro-benzo[1,4]dioxin-6-ylamino)-6,7-diethoxy-quinoline-3-carbonitrileEXAMPLE 244-(1H-Indazol-6-ylamino)-6,7-bis-(2-methoxy-ethoxy)-quinoline-3-carbonitrileEXAMPLE 254-(1,4-Dioxo-1,2,3,4-tetrahydro-phthalazin-6-ylamino)-6,7-diethoxy-quinoline-3-carbonitrileEXAMPLE 26 6,7-Diethoxy-4-(indan-5-ylamino)-quinoline-3-carbonitrileEXAMPLE 274-(2,4-Dioxo-1,4-dihydro-2H-benzo[d][1,3]oxazin-6-ylamino)-6,7-diethoxy-quinoline-3-carbonitrileEXAMPLE 286,7-Diethoxy-4-(1-oxo-indan-5-ylamino)-quinoline-3-carbonitrile EXAMPLE296,7-Diethoxy-4-(3-oxo-1,3-dihydro-isobenzofuran-5-ylamino)-quinoline-3-carbonitrileEXAMPLE 304-(1,1-Dioxo-1H-1-benzo[b]thiophen-6-ylamino)-6,7-diethoxy-quinoline-3-carbonitrileEXAMPLE 317-Ethoxy-4-(1H-indazol-6-ylamino)-6-methoxy-quinoline-3-carbonitrileEXAMPLE 326-Ethoxy-4-(1H-indazol-6-ylamino)-7-methoxy-quinoline-3-carbonitrileEXAMPLE 336,7-Diethoxy-4-(1-methyl-2,5-dioxo-2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepin-7-ylamino)-quinoline-3-carbonitrileEXAMPLE 344-(1H-Indazol-6-ylamino)-6-methoxy-7-(3-morpholin-4-yl-propoxy)-quinoline-3-carbonitrileEXAMPLE 354-({3-chloro-4-[(1-methyl-1H-imidazol-2-yl)sulfanyl]phenyl}amino)-7-methoxy-6-nitro-3-quinolinecarbonitrileEXAMPLE 366-amino-4-({3-chloro-4-[(1-methyl-1H-imidazol-2-yl)sulfanyl]phenyl}amino)-7-methoxy-3-quinolinecarbonitrileEXAMPLE 37(2E)-N-[4-({3-chloro-4-[(1-methyl-1H-imidazol-2-yl)sulfanyl]phenyl}amino)-3-cyano-7-methoxy-6-quinolinyl]-4-(dimethylamino)-2-butenamideEXAMPLE 384-{[3-chloro-4-(1,3-thiazol-2-ylsulfanyl)phenyl]amino}-7-methoxy-6-nitro-3-quinolinecarbonitrileEXAMPLE 396-amino-4-{[3-chloro-4-(1,3-thiazol-2-ylsulfanyl)phenyl]amino}-7-methoxy-3-quinolinecarbonitrileEXAMPLE 40(2E)-N-(4-{[3-chloro-4-(1,3-thiazol-2-ylsulfanyl)phenyl]amino}-3-cyano-7-methoxy-6-quinolinyl)-4-(dimethylamino)-2-butenamideEXAMPLE 414-[3-chloro-4-(1H-imidazol-1-yl)anilino]-7-methoxy-6-nitro-3-idazol-1-yl)anilino]-7-methoxy-6-nitro-3-quinolinecarbonitrileEXAMPLE 426-amino-4-[3-chloro-4-(1H-imidazol-1-yl)anilino]-7-methoxy-3-4-(1H-imidazol-1-yl)anilino]-7-methoxy-3-quinolinecarbonitrileEXAMPLE 43(E)-N-{4-[3-chloro-4-(1H-imidazol-1-yl)anilino]-3-cyano-7-methoxy-6-quinolinyl}-azol-1-yl)anilino]-4-(dimethylamino)-2-)anilino-2-butenamideEXAMPLE 444-{3-chloro-4-[(4-oxo-3,4-dihydro-2-quinazolinyl)sulfanyl]anilino}-7-methoxy-6-hydro-2-quinazolinyl)sulfanyl]anilino}-7-methoxy-6-nitro-3-quinolinecarbonitrileEXAMPLE 456-amino-4-{3-chloro-4-[(4-oxo-3,4-dihydro-2-quinazolinyl)sulfanyl]anilino}-7-3,4-dihydro-2-quinazolinyl)sulfanyl]anilino}-7-methoxy-3-quinolinecarbonitrileEXAMPLE 46(E)-N-(4-{3-chloro-4-[(4-oxo-3,4-dihydro-2-quinazolinyl)sulfanyl]anilino}-3-fanyl]anilino}-3-cyano-7-methoxy-6-quinolinyl)-4-(dimethylamino)-2-butenamideEXAMPLE 47(E)-N-(4-{4-[acetyl(3-pyridinylmethyl)amino]-3-chloroanilino≢-3-cyano-7-methoxy-]-3-chloroanilino}-3-cyano-7-methoxy-6-quinolinyl)-4-(dimethylamino)-2-oanilino}-3-cyano-7-methoxy-6-quinolinyl)-4-(dimethylamino)-2-butenamideEXAMPLE 48N-{2-chloro-4-[(3-cyano-7-methoxy-6-nitro-4-quinolinyl)amino]phenyl}-N-(3-7-methoxy-6-nitro-4-quinolinyl)amino]phenyl}-N-(3-pyridinylmethyl)acetamideEXAMPLE 49N-{4-[(6-amino-3-cyano-7-methoxy-4-quinolinyl)amino]-2-chlorophenyl}-N-(3—methoxy-4-quinolinyl)amino]-2-chlorophenyl}-N-(3-pyridinylmethyl)acetamideEXAMPLE 50N-(4-{[6-(acetylamino)-3-cyano-7-methoxy-4-quinolinyl]amino]-2-chlorophenyl)-N-ano-7-methoxy-4-quinolinyl]amino}-2-chlorophenyl)-N-(3-pyridinylmethyl)acetamideEXAMPLE 514-{3-chloro-4-[(1-methyl-1H-imidazol-2-yl)sulfanyl]anilino}-6-methoxy-7-[3-(4-morpholinyl)propoxy]-3-quinolinecarbonitriteEXAMPLE 524-(3-chloro-4-{[5-(trifluoromethyl)-1,3,4-thiadiazol-2-yl]amino}anilino)-7-ethoxy-6-nitro-3-quinolinecarbonitrileEXAMPLE 53(E)-N-[4-(3-chloro-4-{[5-(trifluoromethyl)-1,3,4-thiadiazol-2-yl]amino}anilino)-3-cyano-7-ethoxy-6-quinolinyl]-4-(dimethylamino)-2-butenamideEXAMPLE 544-[3-chloro-4-(4-pyridinyloxy)anilino]-7-ethoxy-6-nitro-3-quinolinecarbonitrileEXAMPLE 556-amino-4-[3-chloro-4-(4-pyridinyloxy)anilino]-7-ethoxy-3-quinolinecarbonitrileEXAMPLE 56(E)-N-{4-[3-chloro-4-(4-pyridinyloxy)anilino]-3-cyano-7-ethoxy-6-quinolinyl}-4-(dimethylamino)-2-butenamideEXAMPLE 574-{3-chloro-4-[(3-pyridinylmethyl)amino]anilino}-7-methoxy-6-nitro-3-quinolinecarbonitrileEXAMPLE 58(E)-N-(4-{3-chloro-4-[(4-phenyl-1,3-thiazol-2-yl)sulfanyl]anilino}-3-cyano-7-methoxy-6-quinolinyl)-4-(dimethylamino)-2-butenamideEXAMPLE 596-amino-4-(3-chloro-4-{[5-(trifluoromethyl)-1,3,4-thiadiazol-2-yl]amino}anilino)-7-ethoxy-3-quinolinecarbonitrileEXAMPLE 604-[3-chloro-4-(1H-imidazol-1-ylmethyl)anilino]-7-ethoxy-6-nitro-3-quinolinecarbonitrileEXAMPLE 616-amino-4-[3-chloro-4-(1H-imidazol-1-ylmethyl)anilino]-7-ethoxy-3-quinolinecarbonitrileEXAMPLE 62(E)-N-{4-[3-chloro-4-(1H-imidazol-1-ylmethyl)anilino]-3-cyano-7-ethoxy-6-quinolinyl}-4-(dimethylamino)-2-butenamideEXAMPLE 634-{3-chloro-4-[(4-methyl-2-pyrimidinyl)sulfanyl]anilino}-7-ethoxy-6-nitro-3-quinolinecarbonitrileEXAMPLE 646-amino-4-{3-chloro-4-[(4-methyl-2-pyrimidinyl)sulfanyl]anilino}-7-ethoxy-3-quinolinecarbonitrileEXAMPLE 65(E)-N-(4-{3-chloro-4-[(4-methyl-2-pyrimidinyl)sulfanyl]anilino}-3-cyano-7-ethoxy-6-quinolinyl)-4-(dimethylamino)-2-butenamideEXAMPLE 66(E)-N-(4-{3-chloro-4-[(4,6-dimethyl-2-pyrimidinyl)sulfanyl]anilino}-3-cyano-7-ethoxy-6-quinolinyl)-4-(dimethylamino)-2-butenamideEXAMPLE 677-ethoxy-6-nitro-4-[4-[(4-phenyl-1,3-thiazol-2-yl)sulfanyl]-3-(trifluoromethyl)anilino]-3-quinolinecarbonitrileEXAMPLE 686-Amino-7-ethoxy-4-[4-(4-phenyl-thiazol-2-ylsulfanyl)-3-trifluoromethyl-phenylamino]-quinoline-3-carbonitrileEXAMPLE 69(E)-N-{3-cyano-7-ethoxy-4-[4-[(4-phenyl-1,3-thiazol-2-yl)sulfanyl]-3-(trifluoromethyl)anilino]-6-quinolinyl}-4-(dimethylamino)-2-butenamideEXAMPLE 70(E)-N-(4-{3-chloro-4-[(5-phenyl-1,3-thiazol-2-yl)sulfanyl]anilino}-3-cyano-7-methoxy-6-quinolinyl)-4-(dimethylamino)-2-butenamideEXAMPLE 71(E)-N-{4-[3-chloro-4-(1,3-thiazol-2-ylsulfanyl)anilino]-3-cyano-7-methoxy-6-quinolinyl}-4-[(2-methoxyethyl)(methyl)amino]-2-butenamide

(E)-N-{4-[3-chloro-4-(1,3-thiazol-2-ylsulfanyl)anilino]-3-cyano-7-methoxy-6-quinolinyl}-4-[(2-methoxyethyl)(methyl)amino]-2-butenamidewas prepared by adding dropwise, 3.43 g (18.71 mmol, 1.95 mL)4-bromocrotonyl chloride in 12 mL THF over 45 minutes to a stirredsolution of 4.7 g (10.69 mmol)6-amino-4-[3-chloro-4-(thiazol-2-ylsulfanyl)-phenylamino]-7-methoxy-quinolin-3-carbonitrilein 588 mL THF containing 3.73 mL (21.36 mmol) diisopropylethylamine, at0° C. under nitrogen. The reaction produced a mixture of 4-bromo-(andchloro)-but-2-enoic acid{4-[3-chloro-4-(thiazol-2-ylsulfanyl)-phenylamino]-3-cyano-7-methoxy-quinolin-6-yl}-amide.A 300 mL portion of the solution was cooled to 0° C. and 2.38 g (26.7mmol) (2-methoxyethyl)-methylamine in 11 mL THF was added dropwise.After the reaction had warmed to room temperature, 401 mg (0.5 eq) ofsodium iodide was added and the solution was stirred overnight. Thesolvents were evaporated to leave a red gum, which was partitionedbetween EtOAc and saturated NaHCO₃. After standing overnight, the layerswere separated and the organic layer was dried and evaporated.Chromatography of the residue on a short column of Kieselgel 60, elutingwith EtOAc, then EtOAc/15% MeOH, and finally EtOAc/15% MeOH/1% Et₃Nyielded 1.3 g (41%) of the product as a yellow glass; HRMS (ESI) m/z595.13338 (M)⁺¹, Δ=−2.28 mmu.

EXAMPLE 72(E)-N-(4-{3-chloro-4-[(4-phenyl-1,3-thiazol-2-yl)sulfanyl]anilino}-3-cyano-7-ethoxy-6-quinolinyl)-4-(dimethylamino)-2-butenamideEXAMPLE 734-{3-chloro-4-[(1-methyl-1H-imidazol-2-yl)sulfanyl]anilino}-6-methoxy-7-[3-(1H-1,2,3-triazol-1-yl)propoxy]-3-quinolinecarbonitrileEXAMPLE 744-{3-chloro-4-[(4,6-dimethyl-2-pyrimidinyl)sulfanyl]anilino}-7-ethoxy-6-nitro-3-quinolinecarbonitrileEXAMPLE 756-amino-4-[3-chloro-4-[(4,6-dimethyl-2-pyrimidinyl)sulfanyl]anilino}-7-ethoxy-3-quinolinecarbonitrileEXAMPLE 76(2E)-N-{4-[3-chloro-4-(2-thienylmethoxy)anilino]-3-cyano-7-ethoxy-6-quinolinyl}-4-(dimethylamino)-2-butenamide

Representative compounds of this invention were evaluated in severalstandard pharmacological test procedures that showed that the compoundsof this invention possess significant activity as inhibitors of HER-2and are antiproliferative agents. Based on the activity shown in thestandard pharmacological test procedures, the compounds of thisinvention are therefore useful as antineoplastic agents. The testprocedures used and results obtained are shown below.

Kinase Assays: example 1, example 2 and example 3 are potent inhibitorsof the HER-2 enzyme, example 20 is not. Purified recombinant C-terminalfragment of each enzyme is incubated with ATP in the absence or presenceof a range of compound concentrations. Autophosphorylation of thereceptors was evaluated with phosphotyrosine antibodies in an ELISAformat. In a cell-free autophosphorylation assay using the recombinantcytoplasmic domain of HER-2, all three inhibitors reduced enzymeactivity by 50% (IC₅₀) at concentrations between 33-65 nM (Table 1).TABLE 1 Enzyme IC₅₀ (μg/mL) Compound HER-2 EGFR Example 1 0.036 0.028Example 2 0.033 0.051 Example 3 0.019 0.019 Example 20 0.58 0.02They also inhibited EGFR under similar assay conditions at 33-92 nM.

Cell Proliferation Assays: example 1, example 2, and example 3 repressedthe proliferation of a mouse fibroblast cell line transfected with theHER-2 oncogene (3T31neu) by 50% (IC₅₀) at 3-5 nM (Table 2). This valuewas substantially lower than that obtained with the isogenicuntransfected cells (3T3; IC₅₀ 683-906 nM), indicating a high degree ofselectivity for this oncogenic pathway. Cells were incubated withvarious concentrations of compound for 2 days (6 days for BT474 cells).Cell survival was determined using a protein binding dye assay(SRB),(Rubinstein LV, Shoemaker R H, Paull K D, Simon R M, Tosini S,Skehan P, Scudiero D A, Monks A, Boyd M R. Comparison of in vitroanticancer-drug-screening data generated with a tetrazolium assay versusa protein assay against a diverse panel of human tumor cell lines. J.Natl. Cancer Inst. 82(13):1113-8, 1990, Skehan P, Storeng R, Scudiero D,Monks A, McMahon J, Vistica D, Warren JT, Bokesch H, Kenney S, Boyd MR.New colorimetric cytotoxicity assay for anticancer-drug screening. J.Natl. Cancer Inst. 82(13):1107-12, 1990).

The concentration of drug (nM) which inhibits enzyme activity or cellproliferation by 50% is shown. The three inhibitors also inhibited twoother HER-2 overexpressing breast cancer cell lines, SK-Br-3 and BT474(IC₅₀ 2-4 nM), but were much less active on MDA-MB-435 and SW620 cells(a breast cancer and a colon cancer cell line, respectively), that areEGFR— and HER-2-negative. The compounds repressed the epidermalcarcinoma cell line, A431, that overexpresses EGFR (IC₅₀ 81-120 nM)(Table 2). TABLE 2 CELL IC₅₀ (μg/mL) EGFR − − +++ − + − − Her-2 − +++ ++++ +++ − − COMPOUND 3T3 3T3/NEU A431 SKBr3 BT474 MDA-MB-435 SW620Example 1 0.38 0.0029 0.062 0.0015 0.0014 0.47 0.24 Example 2 0.390.0018 0.045 0.001 0.0013 0.44 0.44 Example 3 0.52 0.0023 0.069 0.00150.0024 0.51 0.29 Example 20 0.26 0.0230 0.030 0.0071 0.020 0.34 0.32Example 21 0.463 0.62 0.01 4.57 1.84 Example 22 0.933 0.123 0.0374 0.3650.286 Example 23 0.375 0.27 0.281 0.235 0.411 Example 24 >5 1.961 >52.045 >5 Example 25 >5 >5 >5 >5 >5 Example 26 0.0198 0.342 0.294 0.3520.294 Example 27 4.616 >5 >5 >5 2.922 Example 28 0.0311 0.0181 0.02810.028 0.0244 Example 29 3.301 >5 >5 3.404 1.565 Example 30 0.251 0.2570.336 0.00328 0.146 Example 31 0.0267 0.0368 0.022 0.0359 0.0212 Example32 4.801 0.786 2.094 2.626 4.313 Example 33 >5 >5 >5 >5 >5 Example34 >5 >5 >5 >5 >5 Example 35 4.09 2.88 0.669 1 1.55 Example 36 1.06 3.040.011 0.39 3.16 Example 37 0.02 0.02 0.0004 0.43 0.43 Example 38 0.2620.148 0.124 0.35 0.15 Example 39 0.333 0.663 0.236 0.65 0.55 Example 400.002 0.017 0.0007 0.33 1.13 Example 41 1.09 1.79 1.48 0.95 1.66 Example42 0.53 1.63 1.73 1.27 5.99 Example 43 1.46 0.51 0.32 0.57 1.45 Example44 4.54 2.28 4.54 >5 1.96 Example 45 1.88 1.22 2.15 4.58 4.66 Example 460.15 0.34 0.06 >5 >5 Example 47 >5 0.646 >5 1.16 1.64 Example 48 1.791.6 0.68 2.61 2.57 Example 49 2.4 >5 3.41 3.76 >5 Example 50 >5 3.683.94 >5 >5 Example 51 0.196 0.775 0.32 2.18 Example 52 1.89 1.79 1.221.84 2.54 Example 53 0.89 0.728 0.179 0.95 1.05 Example 54 >5 >5 >52.54 >5 Example 55 >5 >5 >5 1.61 >5 Example 56 >5 3.27 1.51 2.06 >5Example 57 4.03 1.6 0.726 1.87 3.21 Example 58 0.028 0.162 0.005 0.230.57 Example 59 >5 >5 0.551 0.91 1.38 Example 60 >5 >5 2.44 >5 >5Example 61 >5 >5 0.75 >5 >5 Example 62 0.99 0.95 0.045 2.1 3.8 Example63 1.49 1.2 0.45 1.3 0.9 Example 64 3.03 1.53 >5 1.8 2 Example 65 0.0030.12 0.001 0.3 0.2 Example 66 0.01 0.24 0.006 0.2 0.32 Example 67 0.680.76 0.268 0.4 0.4 Example 68 2.52 >5 0.943 2.5 3.1 Example 69 0.420.3 >5 0.3 0.6 Example 70 0.12 0.22 0.01 0.08 0.5 Example 71 0.002 0.030.002 0.09 0.4 Example 72 0.02 0.24 0.006 0.18 0.54 Example 73 0.9731.83 0.104 3.69 Example 74 >5 >5 >5 4.1 >5 Example 75 2.12 0.76 0.98 1.31.36 Example 76 0.41 0.0039 0.066 0.004 0.003 0.77 0.25

Receptor Phosphorylation: Compounds that repressed the proliferation ofa mouse fibroblast cell line transfected with the HER-2 oncogene(3T3/neu) by 50% (IC₅₀) <0.05 μg/ml in Table 2 above were tested for invitro phosphorylation. For Her-2 and EGFR phosphorylation assays, cells(BT474 and A431, respectively) were incubated with variousconcentrations of compound for 3 hours at 37° C. Protein extracts wereanalyzed by immunoblotting using phospho-tyrosine antibodies. Blots werequantified by densitometric scanning. Concentration of compound (nM)which inhibits phosphorylation by 50% was determined. Example 1, Example3 and HKI-272 decreased ligand-independent receptor phosphorylation by50% (IC₅₀) at 5-23 nM in BT474 cells (Table 3). They also repressedEGF-dependent phosphorylation of EGFR in A431 cells at a comparable dose(IC₅₀ 3-7 nM). TABLE 3 IC50 (μg/mL) Compound BT474 A431 Example 1 0.00750.0031 Example 2 0.0026 0.0014 Example 3 0.013 0.0042 Example 20 0.0800.0031 Example 37 <1 Example 40 10-50 Example 58  50-500 Example 760.0015 0.0025

IN VIVO: The in vivo antitumor activity of example 3 was evaluated intumor xenograft models. Tumor cells (grown in tissue culture) or tumorfragments were implanted subcutaneously in female nude mice. Treatmentwas initiated after tumors had reached a size of 90-200 mg, followingrandom assignment of the animals to different treatment groups(staging). Alternatively (3T3/neu), treatment was initiated the dayafter tumor implantation, due to the rapid outgrowth of these tumors.Compounds were formulated in 0.5% Methocel-0.4% polysorbate-80(Tween-80) and administered daily, PO, by gavage. Tumor mass [(L×W²)/2]was determined every 7 days. Statistical significance of compoundeffects was evaluated using Student's t-test.

The activity of example 3 was first evaluated in xenografts of 3T3/neucells example 3 inhibited tumor growth when administered to animals at20 mg/kg/day (65% inhibition, day 21), 40 mg/kg/day (97% inhibition),and 80 mg/kg/day (99% inhibition). These results were almost identicalto those obtained with example 2 treatment (53%, 95%, and 98%inhibition, respectively at 20, 40 and 80 mg/kg/day). In two otherindependent tests, EXAMPLE 3 treatment produced astatistically-significant inhibition of tumor growth (21-33%) at a doseof 10 mg/kg/day. Based on these studies, the minimum efficacious dose(MED) was estimated to be 10 mg/kg/day. This is the smallest dose thatproduces a sustained, statistically-significant (p<0.05) reduction oftumor growth.

The effect of example 3 was next studied in xenografts ofHER-2-dependent human tumor cell lines. In animals bearing BT474xenografts, example 3 treatment reduced tumor growth when dosed between10 mg/kg/day and 40 mg/kg/day. Maximum inhibition was observed on day21, and ranged from 59% (10/mg/kg/day) to 96% (40 mg/kg/day). Forexample 2, inhibition ranged from 76% (10 mg/kg/day) to 95% (40mg/kg/day). Similar results were obtained in two other independentexperiments. In animals bearing xenografts of SUM-190 (a secondHER-2-dependent breast cancer cell line), example 3 treatment resultedin substantial repression of tumor growth when dosed at 40 mg/kg/day(94% inhibition, day 28). Example 3 was also effective againstxenografts of SK-OV-3 (a HER-2-dependent human ovarian carcinoma cellline). Here, example 3 was active between 20 mg/kg/day (86% inhibition,day 35) and 60 mg/kg/day (91% inhibition). The MED in the HER-2overexpressing human xenograft models was estimated at 10 mg/kg/day,similar to example 2. In these studies, there was no decrease in tumorsize below the initial size at the start of dosing. Furthermore, tumorsshowed evidence of re-growth when treatment was completed, which isconsistent with a non-cytotoxic mode of action for example 3.

1. A compound of formula 1 having the structure:

wherein: R₁ is halogen; R₂ is a pyridinyl, thiophene, pyrimidine,thiazole, or phenyl optionally substituted with up to threesubstituents; R₃ is —O— or —S—; R₄ is methyl or CH₂CH₂OCH₃; R₅ is ethylor methyl; and n is 0 or
 1. 2. The compound according to claim 1, whichis:(E)-N-{4-[4-(benzyloxy)-3-chloroanilino]-3-cyano-7-ethoxy-6-quinolinyl}-4-(dimethylamino)-2-butenamideor a pharmaceutically acceptable salt thereof.
 3. The compound accordingto claim 1, which is:(E)-N-{4-[3-chloro-4-(2-pyridinylmethoxy)anilino]-3-cyano-7-ethoxy-6-quinolinyl}-4-(dimethylamino)-2-butenamideor a pharmaceutically acceptable salt thereof.
 4. The compound accordingto claim 1, which is:(E)-N-(4-{3-chloro-4-[(3-fluorobenzyl)oxy]anilino}-3-cyano-7-ethoxy-6-quinolinyl)-4-(dimethylamino)-2-butenamideor a pharmaceutically acceptable salt thereof.
 5. The compound accordingto claim 1, which is:(2E)-N-(4-{[3-chloro-4-(1,3-thiazol-2-ylsulfanyl)phenyl]amino}-3-cyano-7-methoxy-6-quinolinyl)-4-(dimethylamino)-2-butenamideor a pharmaceutically acceptable salt thereof.
 6. The compound accordingto claim 1, which is:(E)-N-(4-{3-chloro-4-[(4,6-di-methyl-2-pyrimidinyl)sulfanyl]anilino}-3-cyano-7-ethoxy-6-quinolinyl)-4-(dimethylamino)-2-butenamideor a pharmaceutically acceptable salt thereof.
 7. A compound comprising(E)-N-{4-[3-chloro-4-(1,3-thiazol-2-ylsulfanyl)anilino]-3-cyano-7-methoxy-6-quinolinyl}-4-[(2-methoxyethyl)(methyl)amino]-2-butenamideor a pharmaceutically acceptable salt thereof.
 8. A method of treating,inhibiting the growth of, or eradicating neoplasms in a mammal in needthereof which comprises administering to said mammal an effective amountof a compound of formula 1 having the structure:

wherein: R₁ is halogen; R₂ is a pyridinyl, thiophene, pyrimidine,thiazole, or phenyl optionally substituted with up to threesubstituents; R₃ is—or —S—; R₄ is methyl or CH₂CH₂OCH₃; R₅ is ethyl ormethyl; and n is 0 or
 1. 9. The method according to claim 8, wherein thecompound is:(E)-N-{4-[4-(benzyloxy)-3-chloroanilino]-3-cyano-7-ethoxy-6-quinolinyl}-4-(dimethylamino)-2-butenamideor a pharmaceutically acceptable salt thereof.
 10. The method accordingto claim 8, wherein the compound is:(E)-N-{4-[3-chloro-4-(2-pyridinylmethoxy)anilino]-3-cyano-7-ethoxy-6-quinolinyl}-4-(dimethylamino)-2-butenamideor a pharmaceutically acceptable salt thereof.
 11. The method accordingto claim 8, wherein the compound is:(E)-N-(4-{3-chloro-4-[(3-fluorobenzyl)oxy]anilino}-3-cyano-7-ethoxy-6-quinolinyl)-4-(dimethylamino)-2-butenamideor a pharmaceutically acceptable salt thereof.
 12. The method accordingto claim 8, wherein the compound is:(2E)-N-(4-{[3-chloro-4-(1,3-thiazol-2-ylsulfanyl)phenyl]amino}-3-cyano-7-methoxy-6-quinolinyl)-4-(dimethylamino)-2-butenamideor a pharmaceutically acceptable salt thereof.
 13. The method accordingto claim 8, wherein the compound is:(E)-N-(4-{3-chloro-4-[(4-methyl-2-pyrimidinyl)sulfanyl]anilino}-3-cyano-7-ethoxy-6-quinolinyl)-4-(dimethylamino)-2-butenamideor a pharmaceutically acceptable salt thereof.
 14. A method of treating,inhibiting the growth of, or eradicating neoplasms in a mammal in needthereof which comprises administering to said mammal an effective amountof(E)-N-{4-[3-chloro-4-(1,3-thiazol-2-ylsulfanyl)anilino]-3-cyano-7-methoxy-6-quinolinyl}-4-[(2-methoxyethyl)(methyl)amino]-2-butenamideor a pharmaceutically acceptable salt thereof.
 15. The method accordingto claim 14 wherein the neoplasm is selected from the group consistingof breast, kidney, bladder, mouth, larynx, esophagus, stomach, colon,ovary, pancreatic, brain, prostrate, and lung.
 16. A pharmaceuticalcomposition which comprises a compound of formula 1 having the structure

wherein: R₁ is halogen; R₂ is a pyridinyl, thiophene, pyrimidine,thiazole, or phenyl optionally substituted with up to threesubstituents; R₃ is —or —S—; R₄ is methyl or CH₂CH₂OCH₃; R₅ is ethyl ormethyl; and n is 0 or
 1. 17. The pharmaceutical composition according toclaim 16, which is:(E)-N-{4-[4-(benzyloxy)-3-chloroanilino]-3-cyano-7-ethoxy-6-quinolinyl}-4-(dimethylamino)-2-butenamideor a pharmaceutically acceptable salt thereof.
 18. The pharmaceuticalcomposition according to claim 16, which is:(E)-N-{4-[3-chloro-4-(2-pyridinylmethoxy)anilino]-3-cyano-7-ethoxy-6-quinolinyl}-4-(dimethylamino)-2-butenamideor a pharmaceutically acceptable salt thereof.
 19. The pharmaceuticalcomposition according to claim 16, which is:(E)-N-(4-{3-chloro-4-[(3-fluorobenzyl)oxy]anilino}-3-cyano-7-ethoxy-6-quinolinyl)-4-(dimethylamino)-2-butenamideor a pharmaceutically acceptable salt thereof.
 20. The pharmaceuticalcomposition according to claim 16, which is:(E)-N-(4-{3-chloro-4-[(4-methyl-2-pyrimidinyl)sulfanyl]anilino}-3-cyano-7-ethoxy-6-quinolinyl)-4-(dimethylamino)-2-butenamideor a pharmaceutically acceptable salt thereof.
 21. A pharmaceuticalcomposition comprising(E)-N-{4-[3-chloro-4-(1,3-thiazol-2-ylsulfanyl)anilino]-3-cyano-7-methoxy-6-quinolinyl}-4-[(2-methoxyethyl)(methyl)amino]-2-butenamideor a pharmaceutically acceptable salt thereof.
 22. A method ofinhibiting EGFR in a mammal which comprises administering to said mammalan effective amount of a compound of formula 1 having the structure:

wherein: R₁ is halogen; R₂ is a pyridinyl, thiophene, pyrimidine,thiazole, or phenyl optionally substituted with up to threesubstituents; R₃ is —O— or —S—; R₄ is methyl or CH₂CH₂OCH₃; R₅ is ethylor methyl; and n is 0 or
 1. 23. The method according to claim 22,wherein the compound is:(E)-N-{4-[4-(benzyloxy)-3-chloroanilino]-3-cyano-7-ethoxy-6-quinolinyl}-4-(dimethylamino)-2-butenamideor a pharmaceutically acceptable salt thereof.
 24. The method accordingto claim 22, wherein the compound is:(E)-N-{4-[3-chloro-4-(2-pyridinylmethoxy)anilino]-3-cyano-7-ethoxy-6-quinolinyl}-4-(dimethylamino)-2-butenamideor a pharmaceutically acceptable salt thereof.
 25. The method accordingto claim 22, wherein the compound is:(E)-N-(4-{3-chloro-4-[(3-fluorobenzyl)oxy]anilino}-3-cyano-7-ethoxy-6-quinolinyl)-4-(dimethylamino)-2-butenamideor a pharmaceutically acceptable salt thereof.
 26. The method accordingto claim 22, wherein the compound is:(2E)-N-(4-{[3-chloro-4-(1,3-thiazol-2-ylsulfanyl)phenyl]amino}-3-cyano-7-methoxy-6-quinolinyl)-4-(dimethylamino)-2-butenamideor a pharmaceutically acceptable salt thereof.
 27. The method accordingto claim 22, wherein the compound is:(E)-N-(4-{3-chloro-4-[(4-methyl-2-pyrimidinyl)sulfanyl]anilino}-3-cyano-7-ethoxy-6-quinolinyl)-4-(dimethylamino)-2-butenamideor a pharmaceutically acceptable salt thereof.
 28. A method ofinhibiting Her-2 in a mammal which comprises administering to saidmammal an effective amount of a compound of formula 1 having thestructure:

wherein: R₁ is halogen; R₂ is a pyridinyl, thiophene, pyrimidine,thiazole, or phenyl optionally substituted with up to threesubstituents; R₃ is —or —S—; R₄ is methyl- or CH₂CH₂OCH₃; R₅ is ethyl ormethyl; and n is 0 or
 1. 29. The method according to claim 28, whereinthe compound is:(E)-N-{4-[4-(benzyloxy)-3-chloroanilino]-3-cyano-7-ethoxy-6-quinolinyl}-4-(dimethylamino)-2-butenamideor a pharmaceutically acceptable salt thereof.
 30. The method accordingto claim 28, wherein the compound is:(E)-N-{4-[3-chloro-4-(2-pyridinylmethoxy)anilino]-3-cyano-7-ethoxy-6-quinolinyl}-4-(dimethylamino)-2-butenamideor a pharmaceutically acceptable salt thereof.
 31. The method accordingto claim 28, wherein the compound is:(E)-N-(4-{3-chloro-4-[(3-fluorobenzyl)oxy]anilino}-3-cyano-7-ethoxy-6-quinolinyl)-4-(dimethylamino)-2-butenamideor a pharmaceutically acceptable salt thereof.
 32. The method accordingto claim 28, wherein the compound is:(2E)-N-(4-{[3-chloro-4-(1,3-thiazol-2-ylsulfanyl)phenyl]amino}-3-cyano-7-methoxy-6-quinolinyl)-4-(dimethylamino)-2-butenamideor a pharmaceutically acceptable salt thereof.
 33. The method accordingto claim 28, wherein the compound is:(E)-N-(4-{3-chloro-4-[(4-methyl-2-pyrimidinyl)sulfanyl]anilino}-3-cyano-7-ethoxy-6-quinolinyl)-4-(dimethylamino)-2-butenamideor a pharmaceutically acceptable salt thereof.